1. Field of the Invention
The present invention relates generally to the field of microbiology. More particularly, it concerns bacterial cell engineering.
2. Description of Related Art
In 1892, Richard Pfeiffer introduced the revolutionary concept of bacterial endotoxin in his description of a non-proteinaceous, non-secreted toxin bound to the surface of Vibrio cholerae (Pfeiffer et al., 1892). This toxin, now known as lipopolysaccharide (LPS), is the major surface molecule of Gram-negative bacteria that triggers the host immune response during infection (Poltorak et al., 2000; Raetz et al., 2002). LPS is composed of lipid A, core oligosaccharide, and O-antigen (Raetz et al., 2007). Lipid A is recognized by the innate immune system through the conserved pattern recognition receptor, Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) complex, which initiates a robust signal cascade that leads to production of inflammatory cytokines. This signaling is crucial for detection and clearance of infection, but can be potent enough to result in lethal endotoxic shock (Raetz et al., 2002). Such tremendous immunogenicity makes lipid A an attractive therapeutic tool as an adjuvant, but its toxicity is a major concern.
Efforts have been made to dampen the toxicity of whole bacteria by altering the degree of Lipid A acylation. One approach has been to inactivate lpxM, a gene encoding the acyltransferase responsible for converting lipid A from a penta-acylated to a hexa-acylated species. LpxM mutants are under investigation in the development of meningococcal vaccines, oncolytic Salmonella strains that specifically target tumors, and bacterial strains designed for gene therapy. Other efforts to detoxify cells or outer membrane vesicles have included acyl chain modification by the enzymes PagL or PagP. However, no bacterial strains have been previously generated using a complex combinatorial approach to yield a diverse library of in bacterium linked lipid A moieties and antigens.